Cromolyn, a New Hope for Limited Treatment of Neutrophilic Asthma: a Phase II Randomized Clinical Trial.

Background: In this study, we aimed to determine the effects of cromolyn on the clinical outcomes and neutrophilic inflammation in patients with resistant cough-variant asthma. Materials and Methods: Patients with cough-variant asthma, with normal physical examination and spirometry results, were treated by inhaled corticosteroids, antileukotrienes, antibiotics, and proton-pump inhibitors according to the Global Initiative for Asthma (GINA) guidelines. Seventy patients, who were resistant to these treatments, were enrolled in this double-blind randomized clinical trial. After randomization, eligible subjects received a cromolyn metered dose inhaler (MDI) or a placebo MDI, which was completely similar in appearance to the cromolyn inhaler. The primary outcomes included cough and Asthma Control Test (ACT) score. Results: Based on the findings, cough significantly decreased with cromolyn therapy, compared to the placebo group. Other clinical findings, including dyspnea, sputum production, and nocturnal symptoms, also improved. The ACT score significantly improved to a nearly normal level (23.53±2.25) in the cromolyn group. Moreover, fractional exhaled nitric oxide (FeNO) significantly decreased with cromolyn treatment (14±9.31 ppm after treatment vs. 28.88±27.39 before treatment). The neutrophil count significantly decreased in the cromolyn group (from 44±24.2% before the trial to 34.08±16.7% after the trial), while it increased in the placebo group (from 39.67±26.47% to 56.71±27.22%). Conclusion: Cromolyn improved the clinical findings of resistant cough-variant asthma and could suppress neutrophilic inflammation.


INTRODUCTION
Cough-variant asthma is a type of asthma, characterized by cough without dyspnea, wheezing, or spirometry derangement (1,2). Treatment of this disease is similar to routine asthma (3). Occasionally, physicians encounter asthmatic patients who are resistant to both inhaled corticosteroids (ICS) and antileukotrienes (4).
Further investigation for lung infection and treatment of obscure gastroesophageal reflux disease (GERD) are commonly recommended for these patients (5). The predominant inflammatory cells in cough-variant asthma are eosinophils, which are associated with high fractional exhaled nitric oxide (FeNO) and good response to ICS (6). Neutrophilic asthma is not considered a very rare condition. In a previous study, non-eosinophilic asthma was reported in 47% of patients with mild to moderate asthma, who were not responsive to ICS (9), while neutrophilic asthma (more than 76% of sputum inflammatory cells) was reported in 19% of the subjects (10). Neutrophilic asthma tends to be more resistant to ICS and persists for longer periods (11). A recent algorithmic approach to severe asthma considered the treatment of neutrophilic asthma as a clinical problem with no established therapy (12).
Cromolyn (disodium cromoglycate) and nedocromil are two medications with few side effects, which are potential therapeutic substitutes for the resistant form of cough-variant asthma. Although use of these two drugs is no longer recommended in the Global Initiative for Asthma (GINA) guidelines (7,8), they have been recommended in some valid references (13). Moreover, cromolyn has shown good results in children (14). This medication can prevent house-dust induced bronchospasms (15) and effectively relieve exerciseinduced asthma (16).
Experimental studies on the anti-inflammatory effects of chromones have shown that they reduce the accumulation of neutrophils after the induction of animal models by ovalbumin (17

MATERIALS AND METHODS
This study was performed during 2011-2013 and consisted of two phases: 1) screening and pretrial treatment (one year); and 2) a clinical trial with cromolyn and placebo (one year).

Screening Period and Pretrial Phase:
A total of 421 subjects (212 females and 209 males), aged above 15 years, who were referred to a pulmonary subspecialty clinic, were selected to participate in the pretrial phase. The subjects met the following criteria: 1) coughing for more than three weeks without a

Clinical Trial
Groups and random allocation: The participants were randomly divided into two groups. One group received cromolyn inhalers (MDI, Cromolex®, Sina Darou, Tehran, Iran), while the second group received placebo inhalers, which were completely similar to the Cromolex inhalers.

Pretrial Phase
The mean age of the subjects was 43±2 years (range: 7-77 years), and no significant difference was found between males and females (t=0.4, P=0.68). The majority of the participants were housewives (35%) and clerks (22.3%).
None of the subjects reported exposure to heavy air pollution. Comparison of clinical findings between the two groups showed insignificant differences, except for dyspnea, which was more predominant in the cromolyn group (Table 1). ACT results showed an improvement in asthma to almost normal levels (mean ACT score after the trial: 23.53±2.25).
The clinical findings did not show any significant changes in the placebo group, while the ACT score improved significantly (Table 1). However, improvement of ACT score in the cromolyn group was significantly higher than the placebo group. The frequency of all clinical findings was significantly lower in the cromolyn group, compared to the placebo group ( Table 1).
The spirometric parameters did not significantly change in the cromolyn and placebo groups after the trial, except for FEV1/FVC (Table 1). FeNO was mildly elevated in both groups. After the trial, it significantly decreased in the cromolyn group, while no significant change was reported in the placebo group (Table 1).

Inflammatory cells:
Based on the findings, 17% of the subjects in the pretrial phase and 65% of the subjects in the post-trial phase were unable to produce enough sputum for cytological analysis (  (Figure 1).   1). The macrophage and eosinophil percentages were higher in the cromolyn group, but the difference was not significant (17.42±12.2% vs. 7.14±4.52% for lymphocytes and 0.33±0.88% vs. 0.29±0.75% for eosinophils) (Figure 1).  Consequently, antibiotic therapy may improve the outcomes of asthma therapy.
A previous study used clarithromycin as an antibiotic and anti-inflammatory agent for resistant and neutrophilic asthma (24). Although clarithromycin had beneficial effects, we believe that cromolyn is more effective and has fewer side effects. In fact, the frequency of side effects was 12% in clarithromycin treatment and 3% in cromolyn treatment according to our study. It seems that these antibiotic therapies are suitable for more severe cases of asthma, while in patients with cough-variant asthma, it is suggested to treat the patients with cromolyn as a medication without side-effects.
FeNO, as a marker of inflammation, is a useful tool for asthma (6). In a previous study, FeNO in cough-variant asthma was lower than typical asthma (23). In the present study, the cut-off point of FeNO was 28 ppm. Detection of neutrophilic asthma is made by sputum analysis. In our experience, persistent and resistant asthma is the best predictive clinical marker for neutrophilic asthma.
However, the mechanism of action of cromolyn in neutrophilic inflammation is not fully understood.
Mechanisms, such as inhibition of NADPH oxidase and oxygen production in neutrophils (25), inhibition of neutrophil chemotaxis (14), and cell rolling, velocity, adhesion, and migration (26) by reducing intracellular free calcium levels (27) have been introduced.

CONCLUSION
In conclusion, cromolyn could relieve coughing and other symptoms of resistant asthma. It seems that cromolyn is the second most effective drug for neutrophilic asthma after clarithromycin.